As for koji used in production of alcoholic beverages, there are solid koji, which is cultured such that spores of filamentous fungi are inoculated to raw material which has been treated with cooking and the like, and liquid koji, which is cultured such that liquid medium is prepared by adding raw material and other nutrient sources to water, and then spores of koji molds or pre-cultured mycelia of koji molds and the like are inoculate thereto.
In the conventional production of fermented foods and drinks such as alcoholic beverages including, for example, sake, shochu, soy sauce, fermented soybean paste, sweet sake and the like, what is called solid koji which is prepared with the solid culture method has been widely used. The solid culture method is the culture method in which spores of koji molds such as Aspergillus kawachii, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae and the like are dispersed on solid raw material such as steam-cooked cereals to allow koji molds to grow on the solid surface.
For instance, for the production of shochu, the solid koji such as Aspergillus kawachii and Aspergillus awamori have been widely used. However, as the solid culture method is a culture system in which raw materials and koji molds disperse unevenly, it is difficult to make even the factors such as temperature, water content, and various nutrient compositions, and the solid culture method is very complicated in culture control. In addition, the production of koji is often conducted under open conditions, and cares are required in terms of quality control so as to prevent contamination with other bacteria. Therefore, the solid culture method is unsuitable for large-scale production.
In contrast, the liquid culture method is easy to culture control and quality control, so it is suitable for efficient production. However, due to the reason that, for example, enzymatic activity is insufficient for brewing shochu, the culture product obtained by liquid culturing koji molds is rarely used as shochu koji.
In addition to the above-mentioned reasons, a major reason of the culture product obtained with the liquid culture method not being used for producing fermented foods and drinks such as shochu is that the behavior of koji molds to produce enzymes such as amylase and cellulase in the liquid culture is known to be much different from that in the solid culture, and productivity thereof is also known to be poor overall (see Non-Patent Documents 1 and 2).
In production of the alcoholic beverages such as shochu, alcohol is usually generated by simultaneous saccharification and fermentation. Therefore, saccharolytic enzymes from koji molds, which affect supplying glucose to the koji molds, particularly glucoamylase and acid-stable α-amylase are key enzymes in the alcoholic fermentation. However, it is known that activity of glucoamylase is remarkably low in the culture product obtained with the liquid culture method and production behavior thereof is also much different from that in the solid culture (see Non-Patent Documents 3 to 6).
As a method of improving glucoamylase activity of koji molds, there are reported the method of culturing koji molds while giving stresses on the growth of mycelia (see Patent Document 1) and the method of adding roasted cereals to koji mold culture fluid (see Patent Document 2). The method disclosed in Patent Document 1 conducts culture on porous membrane or in inclusive immobilization agent having air gaps to allow expression of the novel gene glaB that encodes glucoamylase, to thereby enhance enzymatic activity. Accordingly, the method requires strict control or specific culture devices, and thus it is not practical. The method disclosed in Patent Document 2 cultures koji molds in liquid medium using roasted cereals as, at least, a portion of the raw material, which requires an additional production step of roasting cereals.
The inventors of the present invention provided an invention related to a method of culturing koji molds using liquid medium containing the saccharides which the koji molds hardly decompose (see Patent Document 3). By liquid culturing koji molds with the invention, a koji mold culture product having high activity of glycolytic enzymes such as glucoamylase, which can be used for producing fermented foods and drinks such as sake, can be obtained conveniently and inexpensively.
On the other hand, recently, the molecular biological analysis on acid-stable α-amylase has been conducted to the details (see Non-Patent Document 7). The analysis has reported as follows: A white koji mold has two different amylase genes which are respectively responsible for two different characteristics, that is, acid-unstable α-amylase and acid-stable α-amylase. The expression behaviors of the respective genes are much different from each other. In liquid culturing, the acid-unstable α-amylase is sufficiently produced, while the acid-stable α-amylase, a key enzyme for brewing shochu is hardly produced.
For producing shochu, brewing is conducted under low-pH environments for preventing the shochu mash from putrefaction. The acid-unstable α-amylase contributes very little to glycolysis in shochu brewing because it is deactivated precisely under low-pH conditions. Therefore, it is indispensable for producing shochu that the acid-stable α-amylase is produced with high yield, which is thought to contribute to the glycolysis in shochu brewing, by liquid culturing koji molds.
The production behavior of acid stable α-amylase in liquid culturing koji molds has been investigated in detail and reported. However, the method uses synthetic medium containing peptone and citrate buffer solution, and requires an culture time of 100 hours or more, so it would be difficult to apply to actual shochu brewing (see Non-Patent Documents 8 to 10).
The inventors of the present invention have already developed a method of producing liquid koji sufficiently having enzymatic activities of glucoamylase and acid-stable α-amylase which are necessary for producing shochu, which involves culturing white koji molds and/or black koji molds in liquid medium containing the cereal of which surface is covered with husks as culture raw material to generate and accumulate simultaneously glucoamylase and acid-stable α-amylase in the culture product. The inventors have succeeded in producing shochu using the liquid koji for the first time (see, for example, the specification of Japanese Patent Application No. 2004-350661).
Meanwhile, there has been conducted investigation on plant fiber degradation enzymes produced with koji in order to additionally improve productivity of sake and shochu. It has been reported that utilization ratio of the raw material of sake mash is improved when plant fiber degradation enzymes such as a cellulolytic enzyme, a xylanolytic enzyme, and a pectolytic enzyme are employed in production of sake (see, Non-patent Documents 11 and 12).
It has been also reported that alcohol yield in shochu is improved by creating the recombinant shochu koji molds to which the cellulolytic enzyme gene of Trichoderma viride is introduced and producing shochu with the recombinant shochu koji molds (see Non-patent Document 13).
As described above, it is widely known that improvement in productivity of the plant fiber degradation enzymes to be used in production of sake or shochu is extremely important for promoting efficiency in production of fermented foods and drinks such as sake or shochu.
However, employing the expensive plant fiber degradation enzyme preparation in production of sake may increase cost, and is not preferable.
In addition, using the recombinant shochu koji mold would let the consumer worries about safety, and is not also preferable.
By the way, ethanol as industrial alcohol is used as raw material for producing foods and drinks such as sweet sake, vinegar and the like, or as raw material for producing industrial chemicals such as flavor, detergent and the like. Further, the ethanol is expected recently to serve as novel energy source which is alternative to a fossil fuel such as petroleum. For instance, investigations and developments of an alcohol fuel such as E3 gasoline which is obtained by mixing gasoline with 3% of ethanol are promoted.
When the industrial alcohol is produced by fermentation method using cereals or tubers as raw material, an enzyme preparation (liquefying enzymes or saccharolytic enzymes) needs to be used for high enzymatic activities (see, for example, Non-patent Document 14).
However, use of the enzyme preparation causes a problem that, in addition to high cost, mashing can not be conducted at high concentration. The mash produced with an enzyme preparation generally has an alcohol content of about 8%. Thus, mashing at higher concentration has been expected for improving productivity.
There may be suggested to use solid koji in which koji molds are grown on the surface of cereals or beans, instead of the enzyme preparation. However, the solid koji is not suitable for large-scale production because it has to be produced in a specific culture mode, that is, solid culture.
On the other hand, liquid koji in which koji molds are cultured in liquid medium is able to control culture easily, so that it is suitable for efficient production.
However, it is known to persons skilled in the art that liquid koji does not provide enzymatic activities required for the alcohol fermentation sufficiently, so there has been no examples where liquid koji is used in actual production.
Non-patent Document 1: Iwashita K. et al: Biosci. Biotechnol. Biochem., 62, 1938-1946 (1998)
Non-patent Document 2: Yuichi Yamane et al.: Journal of the Brewing Society of Japan, 99, 84-92 (2004)
Non-patent Document 3: Hata Y. et al.: J. Ferment. Bioeng., 84, 532-537 (1997)
Non-patent Document 4: Hata Y. et al.: Gene., 207, 127-134 (1998)
Non-patent Document 5: Ishida H. et al.: J. Ferment. Bioeng., 86, 301-307 (1998)
Non-patent Document 6: Ishida H. et al.: Curr. Genet., 37, 373-379 (2000)
Non-patent Document 7: Nagamine K. et al.: Biosci. Biotechnol. Biochem., 67, 2194-2202 (2003)
Non-patent Document 8: Sudo S. et al.: J. Ferment. Bioeng., 76, 105-110 (1993)
Non-patent Document 9: Sudo S. et al.: J. Ferment. Bioeng., 77, 483-489 (1994)
Non-patent Document 10: Shigetoshi Sudo et al.: Journal of the Brewing Society of Japan, 89, 768-774 (1994)
Non-patent Document 11: Yoshizawa et al. Journal of the Brewing Society of Japan, 76, 284-286 (1981)
Non-patent Document 12: Fukuda et al. Journal of Bioscience and Bioengineering, 79, 299-302 (2001)
Non-patent Document 13: Nomachi W. et al., J. Biosci. Bioeng., 93(4), p 382-387, 2002
Non-patent Document 14: Encyclopedia of Brewing, p 352-371, Asakura Publishing, Co., Ltd., first edition issued on Nov. 10, 1988
Patent Document 1: JP 11-225746 A
Patent Document 2: JP 2001-321154 A
Patent Document 3: JP 2003-265165 A